The long-term goal of this proposal is to understand the structural mechanism of the constitutive androstane receptor (CAR). CAR belongs to the nuclear receptor superfamily of proteins. These are hormone-modulated transcription factors that direct almost every aspect of human physiology. CAR is constitutively active in the absence of ligand. Small molecules have been identified that modulate CAR activity. The steroidal molecule, androstanol, functions as an inverse agonist and inhibits the constitutive activity of CAR. Another molecule, TCPOBOP, rescues CAR from the androstanol-mediated repression and can enhance CAR activity. We hypothesize that the CAR structure employs unique, but subtle, modifications from the canonical nuclear receptor structural fold that results in this distinctive behavior. We hypothesize that the three states of CAR, the inactive, androstanol-bound, the active, apo, and the TCPOBOP-bound highly active states are linked by an allosteric mechanism. In Specific Aim 1 we propose to compare the structures of apo CAR with androstenol-bound CAR. In Specific Aim 2, we will compare the CAR:TCPOBOP to apo and androstanol-bound CAR structures. In Specific Aim 3, we will study the mechanism of CAR allosterism by performing structure-based point mutations and binding and activity assays on these mutants. CAR has been shown to direct the transcription of the CYP2 and CYP3 gene products, which belong to the cytochrome P50 class of enzymes. These enzymes are responsible for the metabolism of xenobiotics such as pharmaceutical drugs. CAR has been directly implicated in the metabolism of the analgesic, acetaminophen (commercial nomenclature: Tylenol) other pharmaceutical products. Understanding the CAR molecular mechanism is of great significance in nuclear receptor biology with broad implications in the pharmacology of these proteins. Because of its involvement in metabolism of therapeutics, CAR is itself an attractive target for the design of small molecule ligands that can both enhance or diminish its transcriptional activity.